Oxidative damage, particularly to proteins, has been widely postulated to be a major causative factor in the loss of functional capacity during senescence. The nature of the various mechanisms that may contribute to protein oxidation is only partially understood. In this study, concentrations of two markers for oxidative damage, o,o'-dityrosine and o-tyrosine, were determined using stable isotope dilution gas chromatography-mass spectrometry in four tissues of the mouse, namely heart, skeletal muscle, brain and liver during youth (4 month old), adulthood and old age. A comparison was made between mice that had access to unlimited calories with those that were restricted to 60 % of the caloric intake of the ad libitum regimen. In vitro studies demonstrated that o,o'-dityrosine was generated selectively in proteins exposed to tyrosyl radical. o-Tyrosine increased in proteins oxidized with hydroxyl radical, which also resulted in a variable inc rease in o,o'-dityrosine. In the ad-libitum fed mice, levels of o,o'-dityrosine increased with age in cardiac and skeletal muscle but not in liver or brain. In contrast, o-tyrosine levels did not rise with age in any of the tissues examined. These results suggest that tyrosyl radical-induced protein oxidation increases selectively with age in skeletal muscle and heart. Caloric restriction prevented the increase in o,o'-dityrosine levels in cardiac and skeletal muscle but did not influence o-tyrosine levels in any of the four tissues. This selective increase in o,o'-dityrosine levels and its prevention by life-prolonging caloric restriction regimen raises the possibility that oxidation of muscle proteins by tyrosyl radical contributes to the deterioration of cardiac and skeletal muscle function with advancing age.